High-performance gel permeation chromatography of proteins and peptides on columns of TSK-G2000-SW and TSK-G3000-SW: a volatile solvent giving separation based on charge and size of polypeptides |
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Authors: | G B Irvine C Shaw |
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Institution: | 1. Key Laboratory of Zoonosis of Liaoning Province, College of Animal Science & Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, PR China;2. Research Academy of Animal Husbandry and Veterinary Medicine Sciences of Jilin Province, Changchun 130062, PR China |
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Abstract: | Trifluoroacetic acid (0.1% w/v) is an excellent solvent for polypeptides, is volatile, and has a low absorbance of ultraviolet light of low wavelength. Polypeptides subjected to chromatography on columns of TSK-G2000-SW or TSK-G3000-SW in this solvent were eluted as sharp peaks. Retention volume was dependent not only on molecular weight but also on the number of formal charges per molecule. For polypeptides with a similar molecular weight, that with the highest proportion of basic amino acid residues was eluted earliest. For the TSK-G2000-SW column, the degree of deviation from a linear relationship between elution volume and logarithm of molecular weight was directly proportional to the molecular weight to the power 2/3 divided by the number of positive charges per molecule. Inclusion of 0.25 M sodium chloride in the solvent increased both the upper and lower limits of the molecular weight range over which separation occurred. The use of 0.1% trifluoroacetic acid as the mobile phase is thus particularly applicable to the separation of peptides of low molecular weight. |
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